Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication.

The abuse of paramethoxymethamphetamine (PMMA) and 3,4-methylenedioxymethamphetamine (MDMA) among young people is increasingly serious and has become a public health problem. Since enantiomers of MDMA and PMMA are metabolized at different rates in the body and exhibit different neurotoxicity in tissues, we have developed a simple method for simultaneous enantiomeric determination of PMMA and MDMA, using parallel dual capillary immunoaffinity columns coupled with tandem mass spectrometry. Linear calibration curves were obtained in concentration ranges of 100–1000 ng/mL, with a limit of quantitation of <22 ng/mL. Good interday accuracy and precision were achieved with this method. Besides filtering the urine sample through a 0.45 μm MILLIPORE membrane, no other sample pretreatment was needed, and no toxic organic solvent was used. It is a rapid, environmentally friendly safe method, and could be applied for routine enantiomeric analysis of PMMA and MDMA in the pharmaceutical industry, forensic science, and environmental analysis.

Objective The therapeutic effect of methylphenidate (MPH) in treating attention-deficit/hyperactivity disorder (ADHD) has been related to the alpha-2A adrenergic receptor (ADRA2A) gene -1291C/G single nucleotide polymorphism (SNP). We investigated the effect of MPH in treating Taiwanese children and adolescent with ADHD and its relation to the ADRA2A gene -1291C/G SNP. Methods The subjects with DSM-IV ADHD diagnosis underwent a titration period to find out the dose of MPH for maintenance treatment. After 4 weeks maintenance treatment, the effect of MPH was evaluated by the Swanson, Nolan and Pelham version IV total scores. The subjects with more than 25% score reduction were referred to responders and those with >= 50% improvement were considered as better responders. The -1291C/G variant of the ADRA2A gene was identified by DNA sequencing and what relevance it has to the MPH response was examined by binary logistic regression analysis. Results Of the 59 subjects, 44 (74.6%) were responsive to MPH treatment and the responsiveness was not shown to be associated with the ADRA2A gene -1291C/G SNP. As the responsive subjects were categorized as moderate responders and better responders and subjected to statistical analysis, the GG homozygotes showed a greater chance to have a better response to MPH treatment than CC homozygotes (p=0.02), with an odds ratio of 32.14 (95% CI=1.64-627.80). Conclusion The ADRA2A gene -1291C/G SNP is associated with the efficacy of MPH for the treatment of ADHD in Taiwanese children and adolescents. The responsive subjects bearing homozygous -1291G allele are more likely to have a better response to MPH treatment.

Background: The rise in substance-use-related problems among Malaysian adolescents called for a study to assess the efficacy of an anti-drug educational program to improve the knowledge, attitude and perception (KAP) of Malaysian students regarding substance use. Methods: This study involved 573 students from four Malaysian Chinese schools in Malacca. Each student filled out a Likert-scale mandarin translated questionnaire (KAP-C) on substance use ( Pre-test). Following an educational program on substance use, the KAP-C questionnaire was given again to the students, immediately following the program (PT 1), and after a month (PT 2) and two months (PT3) of the programme. Results: The students showed improvement in knowledge of the types of substance use in PT 1 (p<0.001), PT 2 (p<0.001) and PT 3 (p<0.001). The study also showed persistent changes in their attitude towards drug treatment strategies at two months following the program (p < 0.05). Changes were also noted in their attitudes towards harm reduction strategies. Perceptions towards the substance-related problems changed following the module (p=0.001), and remained even after two months (p<0.001). Conclusion: This module effectively changed the KAP of students on substance use.

A simple and rapid method for direct quantitation of drugs in human urine samples was developed using a system composed of an automatic column switch and two home-made capillary immunoaffinity columns (CIACs, 100 μm × 15 cm).

The abuse of ketamine and amphetamine analogs is associated with incidence of hypertension and strokes involving activation of sympathetic activities. Large cerebral arteries at the base of the brain from several species receive dense sympathetic innervation which upon activation causes parasympathetic-nitrergic vasodilation with increased regional blood flow via axo-axonal interaction mechanism, serving as a protective mechanism to meet O2 demand in an acutely stressful situation. The present study was designed to examine effects of ketamine and amphetamine analogs on axo-axonal interaction-mediated neurogenic nitrergic vasodilation in porcine basilar arteries using techniques of blood-vessel myography, patch clamp and two-electrode voltage clamp, and calcium imaging. In U46619-contracted basilar arterial rings, nicotine (100μM) and electrical depolarization of nitrergic nerves by transmural nerve stimulation (TNS, 8Hz) elicited neurogenic nitrergic vasodilations. Ketamine and amphetamine analogs concentration-dependently inhibited nicotine-induced parasympathetic-nitrergic vasodilation without affecting that induced by TNS, nitroprusside or isoproterenol. Ketamine and amphetamine analogs also concentration-dependently blocked nicotine-induced inward currents in Xenopus oocytes expressing α3β2-nicotinic acetylcholine receptors (nAChRs), and nicotine-induced inward currents as well as calcium influxes in rat superior cervical ganglion neurons. The potency in inhibiting both inward-currents and calcium influxes is ketamine>methamphetamine>hydroxyamphetamine. These results indicate that ketamine and amphetamine analogs, by blocking nAChRs located on cerebral perivascular sympathetic nerves, reduce nicotine-induced, axo-axonal interaction mechanism-mediated neurogenic dilation of the basilar arteries. Chronic abuse of these drugs, therefore, may interfere with normal sympathetic-parasympathetic interaction mechanism resulting in diminished neurogenic vasodilation and, possibly, normal blood flow in the brainstem.

Objective The efficacy of clozapine clearance has been shown to be associated with smoking and genetic polymorphism of CYP1A2. This study aims to investigate the effect of smoking on the plasma level of clozapine in Taiwanese schizophrenic patients and its relevance to the CYP1A2 gene −163A/C single nucleotide polymorphism. Materials and methods A total of 143 hospitalized schizophrenic patients who had received clozapine therapy for at least 14 days were enrolled in this study. The trough plasma concentration of clozapine was measured with LC/MS/MS. The −163A/C variant in the CYP1A2 gene was identified by DNA sequencing and restriction fragment length polymorphism analysis. The effect of smoking on the clozapine level was examined by multiple linear regression analysis and its relation to the −163A/C variant of the CYP1A2 gene was analyzed using a general linear model with Bonferroni correction. Results Patients with smoking habits showed a significantly lower plasma level of clozapine than those without smoking habits (P=0.022) and the difference in clozapine levels between smokers and nonsmokers appeared to be significant in the individuals carrying the homozygous −163A allele (P=0.02). It was also found that nonsmokers carrying the −163A allele tended to have higher plasma levels of clozapine. This tendency was not found in the individuals with smoking habits. Conclusion Cigarette smoking has a significant impact on the plasma level of clozapine in Taiwanese schizophrenic patients carrying the homozygous −163A allele in the CYP1A2 gene. Cigarette smoking may increase the clearance of clozapine in these patients.

A screening procedure was developed for the simultaneous detection of mephedrone, six amphetamine-type stimulants (ATS), ketamine and its two metabolites with electrospray ionization flow injection analysis tandem mass spectrometry (FIA-MS-MS). Urine samples were fortified with deuterated analogues as internal standards, extracted with ethyl acetate and analyzed with FIA-MS-MS. The mass analyzer was operated in multiple reaction monitoring mode. Two product ions were monitored for each drug and internal standards. For each analyte, the limit of detection was less than 4 µg/L, within-day and between-day precisions (percent coefficient of variation) at three different concentrations were less than 7.3% and bias was between -17.3 and 11.8%. Total analysis time with FIA-MS-MS is 1.8 min per sample. A group of 215 urine samples were screened with immunoassay for ATS and analyzed with FIA-MS-MS and gas chromatography-mass spectrometry (GC-MS) for ketamines and ATS. The analysis of ATS by immunoassay and GC-MS was 96.7% concordant. The analysis of three ketamines and seven ATS by FIA-MS-MS and GC-MS was 97.2% concordant. The FIA-MS-MS procedure is efficient, accurate, flexible and capable of detecting analytes of different chemical groups. It can replace immunoassays for the screening of new designer drugs when commercial immunoassays are unavailable.

A convenient synthesis of ketamine metabolite dehydronorketamine-d(4), starting from commercially available deuterium labeled bromochlorobenzene, was achieved. Key steps include Grignard reaction, regioselective hydroxybromination, Staudinger reduction, and dehydrohalogenation.

Designer drugs are introduced periodically to avoid detection and to provide new drugs with different pharmacological activities. During our routine analysis of amphetamine in urine samples, we observed one sample that reacted with immunoassay with high activity. There is one prominent peak in the gas chromatography- mass spectrometry (GC-MS) chromatogram. However, no amphetamine, methamphetamine, MDA, MDMA, MDEA, or ephedrine was detected with GC-MS. Careful examination of the mass spectrum indicated the presence of one fragment ion (m/z 140), which is similar to the base peak of trifluoroacetic anhydride derivative of amphetamine. The characteristic ion cluster representing the presence of one chlorine atom was observed. Investigation with liquid chromatography (LC)-MS detected an unknown compound with molecular ion of m/z 170. This compound was tentatively identified as chloroamphetamine. Pure standard material of p-chloroamphetamine (PCA) was purchased and analyzed with both GC-MS and LC-MS. Identical GC-MS spectra and LC-MS-MS fragmentation patterns were obtained. A GC-MS procedure was developed for the quantitation of PCA. The limits of detection and quantification were 10 μg/L. Precision was between 1.26% and 4.26%, and bias was between -0.91% and 4.27%. The prevalence PCA positive rate is 0.35% of the samples screened positive for amphetamine.

A screening method based on electrospray tandem mass spectrometry (MS-MS) was developed. Urine samples were spiked with ketamine-d(4) and norketamine-d(4) as internal standard and extracted with 0.5 mL ethyl acetate. Extracted samples were monitored with triple-quadrupole MS-MS. Total analysis time was 1.5 min/sample. Limit of detection was 0.1 ng/mL for ketamine, norketamine, and dehydronorketamine (DHNK). Carryover rate was less than 0.06%. Within-run and between-run precision for ketamine, norketamine, and DHNK at three different concentrations (40, 75, and 125 ng/mL) was between 2.1 and 8.2%. Within-run and between-run accuracy, presented as % bias, was between -5.9 and 2.7%. A group of 76 urine samples were screened with ELISA and gas chromatography-mass spectrometry (GC-MS). With GC-MS as the reference method, when ketamine, norketamine, and DHNK were monitored at cutoff concentration of 100 ng/mL, there were 21 positive, 45 negative, 7 false-negative, and 3 false-positive results. A group of 243 samples was screened with MS-MS and analyzed with GC-MS; there were 74 positive, 163 negative, 6 false-positive, and no false-negative results. In conclusion, the MS-MS procedure is accurate, efficient, and suitable for use as a high-throughput screening method for ketamine and metabolites.

The use of gas chromatography-mass spectrometry (GC-MS) in forensic analysis is increasing. To exploit fully the capabilities of MS, labeled standards, that can be used to improve the performance of the quantitative analysis, and to increase accuracy and precision, are required. A series of deuterated internal standards, corresponding to the 2C-series of phenethylamine derivatives, including 4-bromo-2,5-dimethoxyphenethylamine-d6 (2C-B), 4-chloro- 2,5-dimethoxyphenethylamine-d6 (2C-C), 4-iodo-2,5-dimethoxy-phenethylamine-d6 (2C-I), 4-ethylthio-2,5-dimethoxy-phenethylamine-d6 (2C-T-2) and 2,5-dimethoxy-4-n-propylthiophenethylamine-d6 (2C-T-7), were synthesized. These deuterated compounds were used to analyze for the corresponding unlabeled compounds in urine. The analysis was performed using GC-MS, with the selected ion monitoring (SIM) technique, whereby good results were achieved.

DMA, DOB and PMA are increasingly abused central nervous system stimulants with neurotoxic properties. In recent years, many controlled substance analogs (designer drugs) with a large variety of structures have reached the illegal market, making their identification difficult. Therefore, this work studies the synthesis of DOB-d(6), DMA-d(6) and PMA-d(3) as internal standards for use in gas chromatography-mass spectrometry (GC-MS) to identify controlled substances.

A method using an immunoaffinity column (IAC) and liquid chromatography-electrospray ionization mass spectrometry (LC/MS) for on-line detecting the presence of MA in the effluent was developed for the quantitative and enantiomeric determination of (S)-(+)-methamphetamine (d-MA) in urine. The IAC was made in our laboratory and utilized in the LC/MS to simultaneously extract and separate enantiomers of MA from urine samples. An aqueous ammonium acetate buffer was used as the mobile phase. Urine samples were spiked with racemic deuterated methamphetamine (MA-d14) as internal standard (IS), filtered through a membrane, and injected into the LC/MS without any further pre-treatment. Protonated molecular ion of MA and MA-d(14) (m/z 150 and 164) were isolated and further fragmented, the respective product ions, m/z 119 and 130, were collected for quantitative determination. This is an improvement of our previous method (A.C. Lua, Tsong-Yung Chou, J. Chromatogr. A 967 (2002) 191). In the previous method, MA was separated with HPLC, the efflux was fractionated and each fraction was either determined with an immunoassay or GC/MS. Monitoring of MA in the efflux is tedious and time consuming. Urine samples spiked with different concentrations of d-MA were measured by this method. A linear relationship exists in the 150-1050 ng/mL range, and the detection limit (defined as signal-to-noise ratio 3) of d-MA was determined to be 18 ng/mL. The linearity of the method for d-MA can be described by the equation (Y=1.415 x 10(-3)X+0.034, correlation coefficient: r2=0.999). Within run, accuracy and precision (n=6, relative error: -7.2 to +4.0% and relative standard deviation: 3.8-9.3%) of the method are fairly good.

A method for the simultaneous determination of amphetamines and ketamines (ketamine, norketamine and dehydronorketamine) in urine samples by gas chromatography/mass spectrometry was developed and validated. Urine samples were extracted with organic solvent and derivatized with trifluoroacetic anhydride (TFAA). The limits of detection and limits of quantification for each analyte were lower than 19 and 30 ng/mL, respectively. Within-day and between-day precisions were within 0.5% and 10.6%, respectively. Biases for three levels of control samples were within -10.6% and +7.8%. The concentration of dehydronorketamine was greater than those of ketamine or norketamine in 19 of 35 ketamine-positive samples. A group of 110 human urine samples previously determined to contain at least one of the target analytes was analyzed using the new method, and excellent agreement was observed with previous results.

Objective: The rapid increase of ketamine abuse worldwide has created a need for a fast and efficient screening procedure. Currently, there is no commercially available immunoassay. We developed a simple extraction and a rapid gas chromatography-nitrogen phosphorus detector (GC-NPD) procedure to screen out negative samples. Materials and Methods: Urine samples (1mL) were spiked with chlorpheniramine as an internal standard and extracted with 0.2mL of hexane. Samples were screened for ketamine (K) and its metabolite, norketamine (NK) with GC-NPD without derivatization. Results: The total analysis time was 4 minutes for each sample. The detection limits for K and NK were 5.0 and 5.6ng/mL, respectively. The within-run precisions (% CV) and accuracies (% error) for 80, 240 and 400ng/mL of K and NK were all between -4.00% and 4.75%. A group of 104 urine samples collected from patrons at discos in Taiwan were screened with GC-NPD and confirmed with gas chromatography/mass spectrometry (GC-MS). The total analysis time for GC-MS confirmation was 20 minutes per sample. The sensitivity was 10000 and specificity was 97.6%. Conclusions: A simple and fast procedure was employed (60 samples per hour) to extract ketamine from urine samples for GCNPD screening. Total analysis time was 4 minutes for each sample. The detection limits of K and NK were less than 6ng/mL. The precision (% CV) and accuracy (% error) were less than 500. The GC-NPD screening method is rapid, sensitive and suitable for high volume screening for ketamine.

Objectives: To investigate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) and compare the serologic responses to various GBV-C/HGV markers in eastern Taiwan aborigines.

Immunoaffinity chromatographic columns were prepared for direct enantiomeric determination of racemic methamphetamine and amphetamine in this study. The stationary phase was synthesized by covalently bonding an anti-d-methamphetamine monoclonal antibody onto a pre-activated support (e.g. silica, sepharose 4B). Chromatographic results revealed that the immunoaffinity columns achieved enantiomeric separation of racemic amphetamine and methamphetamine. The immunoaffinity columns also have the ability to directly extract d-methamphetamine from urine by changing the pH of the mobile phase, this ability making it practical for the columns to determine a very low concentration of d-methamphetamine in urine.